map kinase pathways: functions and modulation · 2017-05-25 · tocris scientific review series 2 |...

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Aroon S. Karra and Melanie H. Cobb

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

Dr. Melanie Cobb is a Professor of Pharmacology whose laboratory studies multiple aspects of kinase signaling. Aroon Karra’s research focuses on the connections between the ERK1/2 pathway and chromatin regulation.

Correspondence e-mail: [email protected]

MAP Kinase Pathways: Functions and Modulation

Tocris Scientific Review Series

Background

Mitogen-activated protein kinase (MAPK) networks are criti-cal for the transmission of extracellular signals into appro-priate intracellular responses. Prototypical MAPK activation employs a three-kinase core module consisting of a MAPK kinase kinase (MAPKKK or MAP3K) that phosphorylates and activates a MAPK kinase (MAP2K, MEK, or MKK) that in turn phosphorylates and dramatically increases the activity of one or more MAPKs (Figure 2). Once activated, MAPKs can phosphorylate a wide array of intracellular targets that include cytoskeletal elements, membrane transporters, nuclear pore proteins, transcription factors, as well as other protein kinases.

Discovery of MAPKsThe MAPKs extracellular signal regulated protein kinases 1 and 2 (ERK1/2) were initially identified as mitogen-stimulated ~42kDa phosphoproteins in the late 1980s.1,2 Early studies demonstrated preparations of ERK1/2 retained the ability to phosphorylate the model substrates microtubule-associated protein 2 (MAP2) and myelin basic protein (MBP), and reac-tivate phosphatase-treated ribosomal protein S6 kinase (RSK or p90).3-6

In following years, the MAPK family was discovered to include three c-Jun N-terminal kinases (JNKs), four p38 isoforms, ERK3 isoforms, as well as ERK5 and ERK7 (Figure 2). The first JNK family members were independently identified as cycloheximide-activated MBP kinases and purified for their ability to interact with the N-terminus of the transcription factor c-Jun.7,8 p38α was determined to be an inflammatory cytokine-stimulated tyrosine phosphoprotein, a target of an inhibitor of tumor necrosis factor α (TNFα) production, and a reactivating kinase for MAPK-activated protein kinase 2 (MAPKAP2 or MK2).9-11 PCR-based cloning strategies and a yeast two-hybrid screen identified additional JNK and p38 isoforms, as well as ERKs 5 and 7 (reviewed by Chen et al, Sabapathy, Cuadrado & Nebreda, Nithianandarajah-Jones et al, and Drew et al; a summary of the cellular processes involv-ing these MAPKs is shown in Figure 2, and detailed reviews by Raman et al, Dhillon et al, and Kyriakis & Avruch are also recommended for further reading).12-19 Notably, ERK5 has become a recent target of therapeutic interest20 because of its roles in cancer and structural differences from ERK1 and 2. 21,22 Although similar stimuli affect ERK1/2 and ERK5, particularly growth factors, activation of ERK5 is dependent on different

ContentsBackground .....................................................................................................................1

Discovery of MAPKs ................................................................................................1

Upstream regulation of ERK1/2 ...........................................................2

MAP2Ks .......................................................................................................................2

Raf isoforms and other MAP3Ks .........................................................2

Properties of MAPK cascades ................................................................3

Scaffolding proteins .........................................................................................4

Activation of MAPKs from the cell surface .....................................5

Tyrosine kinase receptor activation of ERK1/2 .......................5

Inhibition of MAPK pathways .........................................................................5

MAPK function studied by inhibition ...............................................5

ERK1/2 pathway inhibitors ....................................................................5

JNK pathway inhibitors .................................................................................6

p38 pathway inhibitors..................................................................................6

Future prospects .........................................................................................................6

References ........................................................................................................................7

Compounds Available from Tocris ..............................................................8

mailto:Aroon.Karra%40utsouthwestern.edu?subject=


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upstream factors.21 Indeed, regulated cross-talk and insulation are both hallmarks of most related MAPK pathways.

Collectively, these findings established that prototypical three-kinase cascades culminating in activation of a MAPK are versatile signaling modules capable of integrating a multitude of inputs into a variety of responses.

Upstream regulation of ERK1/2The observation that Ras and Raf mutations are prevalent in a variety of human tumors served as early evidence for ERK1/2 having roles in proliferation and oncogenic growth.23 Work from multiple laboratories has led to the understanding that Raf isoforms are effectors of Ras small GTP binding proteins and are upstream kinases for MAPK/ERK kinase 1 and 2 (MEK1/2), which in turn activate ERK1/2 via dual phospho-rylation.24 Consistent with these early reports, the capacity to drive tumorigenesis was further demonstrated by the ability of an activated mutant of MEK1 to transform cells and promote tumor growth in nude mice.25 Subsequent work employing dominant interfering mutants, pharmacological inhibitors of MEK1/2, and disruptions in component gene expression all revealed ERK1/2 to be intimately involved in normal processes including embryogenesis, cell differentiation, glucose sensing, and synaptic plasticity.26-31

MAP2KsMAP2Ks are dual-specificity kinases capable of phospho-rylating both tyrosine and serine/threonine residues, and serve as points of signal integration, largely through docking site-mediated protein-protein interactions and the activity of scaffolding proteins. In contrast to MAPKs, which have a wide range of substrates, MAP2Ks are highly specific; they are

primarily (but not exclusively) dedicated to phosphorylation of a small number of MAPK proteins.25

The quintessential MAP2K protein, MEK1, was initially puri-fied as a ~45 kDa activator of ERK1/2, and DNA-based molecu-lar techniques led to the subsequent identification of MEKs 2-7 (reviewed by Chen et al and Lewis et al).12,32-35 While phar-macological inhibition of MEK1/2 is sufficient to effectively shut down ERK1/2 signaling, a recent paper has revealed that MEK1/2 have an additional substrate: the proteotoxic stress response master regulator heat shock factor protein (HSF1).36 In addition to uncovering a connection between MEK1/2 and regulation of proteomic stability, this study also identi-fied opposing roles of ERK1/2 and MEK1/2 within the proteo-toxic stress response, raising the possibility that ERK1/2 and MEK1/2 exert divergent influences on other cellular processes. RNA-sequencing experiments in mouse embryonic stem cells under conditions of ERK1 depletion or pharmacological inhi-bition of MEK1/2 provide some support for the notion that MEK has both ERK-dependent and independent functions, although these experiments are complicated by the contin-ued presence of ERK2.37 Nevertheless, it remains to be seen whether other MAP2Ks also have targets outside their respec-tive MAPKs and, further, how alternative substrates contribute to MAPK signaling regulation.

Raf isoforms and other MAP3KsThe three-kinase cascade, a module conserved from yeast to humans and comprising a MAP3K, MAP2K and a MAPK, is the most readily identifiable feature of MAPK signaling. One of the best-characterized mammalian MAP3Ks is Raf, origi-nally identified as a retroviral oncogene known for its role in cancer promotion. Three isoforms, c-Raf (or Raf-1), B-Raf, and A-Raf, have been identified in mammals.38 In addition to the core ~30 kDa kinase domain, Raf proteins contain an N-terminal regulatory region of roughly the same size that can directly bind Ras. Raf proteins specifically phosphorylate the MAP2Ks MEK1/2 and were initially thought to function in a tissue-specific manner. Subsequent studies, aided by the development of B-Raf inhibitors, led to the understanding that Raf isoforms can dimerize to modulate signal transmission.39,40 The unanticipated actions of these B-Raf inhibitors provoked more in-depth molecular analysis showing that dimerization can enhance Raf activity and that Raf heterodimers display different behaviors.41-43 More recent work suggests that B-Raf dimerization is subject to feedback control by ERK1/2, poten-tially explaining why Raf mutants unable to dimerize maintain inappropriately elevated levels of ERK1/2 signaling.44, 45

Although no direct counterpart to Raf has been found in yeast, the highly conserved nature of MAPK signaling led researchers to look for homologs of the yeast MAP3K Ste11 in mammals. MEKK1 was the first mammalian Ste11 homolog discovered, and despite being able to phosphorylate multiple MAP2Ks in vitro, numerous cell-based studies indicate MEKK1 pre-dominantly coordinates downstream signaling through MEKs

Figure 1 | MAP3K and MAP2K inhibitors

NH2

S

H2N

CN

NC

NH2

S

H2NNH

OH

Br

Br

O

I

U0126 (1144)Potent, selective inhibitor of

MEK1 and 2

GW 5074 (1381)Potent, selective c-Raf1

kinase inhibitor

O

O

O

OHHO

MeO

OHHN

F

IF

F

HN

OHOO

OH

(5Z)-7-Oxozeaenol (3604)Potent and selective TAK1

MAP3K inhibitor

PD 0325901 (4192)Potent MEK1/2 inhibitor

MAP KinASE PAThwAyS: FUnCTionS AnD MoDUlATion

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4 and 7 and the JNK pathway.46-51 Subsequent isolation of related cDNAs have led to the discovery of a family of enzymes (i.e. MEKKs 1-4; ASK1/2; TAK1) that are reviewed by Raman et al and Johnson et al.18,52 As a group, these enzymes display broader substrate specificity than Raf and presumably regulate multiple MAPK pathways in a context-dependent manner.

Two more enzymes that function as MAP3Ks in the ERK1/2 pathway are Mos and Tpl2 (Cot), both originally identified as proteins able to transform cells, and both found to function in specialized situations to activate the core cascade 53,54. Mos is expressed primarily in oocytes where translational recruitment of c-mos mRNA and subsequent MAPK pathway activation is critical for pronuclear formation, while Tpl2 is stabilized in response to lipopolysaccharide.55,56 Another MAP3K family contains homologs of the yeast protein Ste20, TAOs 1-3, which serve as activators of the p38 signaling cascade.18

Figure 2 presents a simplified model of the organization of MAPK cascades with attention drawn to the number of MAP2K-MAPK combinations that a given MAP3K can regulate, and highlights putative nodes of cross-talk.

Properties of MAPK cascadesA large body of work on yeast MAPK cascades and the mam-malian ERK1/2, p38, and JNK signaling pathways has provided insight into the primary features of MAPK core modules, most notably their versatility in processing a wide range of stimuli

into a variety of responses. Efforts to understand how three-kinase cascades achieve such robust signaling have revealed that feedback mechanisms at multiple levels underlie the evolu-tionary success of these pathways. Moreover, multiple biochem-ical mechanisms involving pathway effectors, scaffolds, and targets serve to regulate MAPK signaling dynamics such that one core module can effectively regulate intracellular responses in a context-dependent manner. (For a detailed review on how signaling dynamics can shape cellular responses, refer to Purvis & Lahav.57)

One major feature of ERK1/2 signaling is that two phosphoryla-tion events on the activation loop are required for full activa-tion, the first on a tyrosine residue and a second on a proximal threonine residue. In conjunction with scaffolding proteins that tether the three-kinase cascade together, and owing to its dual-specificity, MEK1/2 can behave either as a single switch via rapid double phosphorylation or can generate a pool of monophosphorylated ERK1/2, thereby increasing the number of contexts that can ultimately achieve full pathway activa-tion.4,58-61 The role of monophosphorylation of MAPKs may not be limited to potentiating a switch-like response to a graded stimulus, as work by Ashwell and colleagues has demonstrated with p38 in T-cells.62 In this system, p38 phosphorylated on the threonine activation site, but not the tyrosine, displays altered substrate specificity, although it is important to note that this mode of activation is specific to T-cells and involves a non-canonical mechanism of p38 activation.

Figure 2 | MAP kinase networks

MAP3K

MAP2K

MAPK

Cellular response

Cascade Yeast

Ste11

Ste7

Fus3

Proliferation

Mammalian

MEK1MEK2

Raf Mos Tpl

ERK1/2

Elk-1

Proliferation,differentiation,

survival,migration,

development

Sap1 bHLH

MAPKAP RSK

MEK4MEK7

JNK1-3

MEK3 MEK6

MEKK TAK ASK TAOMLK

Elk-1 MEF2

MAPKAP

Sap1

RSK

Elk-1

ATF2NFAT4

Jun

Apoptosis,differentiation,metabolism,

inflammation

p38α−δ


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In addition to monophosphorylation, another emerging MAPK property is alternative phosphorylation of a threonine residue proximal to the canonical activation sites described above. In 2009 it was discovered that activated ERK2 can auto-phosphorylate upon dimerization resulting in the promotion of a distinct gene expression program in a model of cardiac

hypertrophy. Subsequent biochemical characterization of a phosphomimetic mutant of the alternative phosphorylation site suggested divergent binding properties of ERK2 modified in that manner.63,64 Although it is far from clear how differ-ent modes of phosphorylation contribute to pathway feedback regulation, these activities likely play some part in conferring network robustness.

Free ERK1/2 can enter and exit the nucleus even in the absence of pathway activation because of its interactions with nuclear pore proteins.65,66 However, full activation of ERK1/2 can pro-mote dimerization and nuclear localization.67 Recently, a small molecule, DEL 22379, able to interrupt dimer formation even when the pathway is activated has been identified, and inter-estingly, inhibition of dimerization seems to alter cytoplasmic ERK1/2 signaling predominantly by disrupting interactions between ERK dimers and scaffold proteins.68,69 More work is required to delineate how this drug affects the extensive nuclear activities of ERK1/2, especially given the potential role of dimerization in driving specific gene expression programs in an ERK1/2-dependent manner.63

Once doubly phosphorylated, ERK1/2 have multiple substrates within the core-signaling pathway. As previously mentioned, ERK1/2 can phosphorylate Raf isoforms to inhibit reactivation by Ras and subsequent phosphorylation of MEK1/2, simulta-neously buffering against inappropriate pathway activation and ensuring a linear response to stimuli.70-75 In yeast it has been demonstrated that the p38 homolog Hog1p phosphor-ylates the signaling scaffold Ste20 (homologous to mammalian PAK) progressively over time, and the authors of a recent study implicated Ste20 in both negative and positive feedback loops because of its ability to act as a rheostatic effector of the MAPK pathway.76

Scaffolding proteinsAlthough the importance of scaffold proteins has been appreci-ated for some time, recent advances in computer-aided mod-eling, in combination with biochemical work, is shedding light on how these proteins exert major influence on cascade func-tion and strongly impact the activities and outputs of MAPK pathways.77 One of the best examples of scaffold-mediated signal specificity is the yeast protein Ste5 in concert with the MAP3K Ste11, which is required to activate the downstream MAPKs Kss1 and Fus3 via Ste7 in response to pheromone.78-81 However, in response to osmotic stress, the sensor protein Sho11 enables Pbs2, the MAP2K upstream of Hog1p, to form a stable interaction with Ste5 and Ste11, thus funneling distinct stimuli through the same signaling components (Figure 3). More recently, a synthetic approach was taken to “rewire” Ste5 with highly modular PDZ domains, in order to better under-stand the effects of scaffolds on pathway dynamics.82 Consistent with previous observations, the authors of this study found that scaffolds can act as logic gates by tethering core components together, as well as modulate pathway output through the recruitment of protein phosphatases.83,84

Table 1 | MAPK pathway components

Gene Aliases

MAPK

ERK2 MAPK1 p42, p41

ERK1 MAPK3 p44, p43

ERK3α MAPK6 rERK3, p97, p63

ERK3β MAPK4 hERK3, ERK3-related, ERK4

JnK1 MAPK8 SAPK1, SAPKγ

JnK2 MAPK9 SAPKα, p54

JnK3 MAPK10 SAPKβ

p38α MAPK14 CSBP, Mxi2 (alternative splice form)

p38β MAPK11 SAPK2, p38-2

p38γ MAPK12 ERK6, SAPK3

p38δ MAPK13 SAPK4

ERK5 MAPK7 BMK1

ERK7 MAPK15 ERK8

MAP2K

Mek1 MAP2K1 Mkk1, MAPKK1

Mek2 MAP2K2 Mkk2

Mek3 MAP2K3 Mkk3

Mek4 MAP2K4 Mkk4, Sek1

Mek5 MAP2K5

Mek6 MAP2K6

Mek7 MAP2K7

MAP3K

C-Raf RAF1

B-Raf BRAF

A-Raf ARAF

Mekk1 MAP3K1 MAPKKK1

Mekk2 MAP3K2

Mekk3 MAP3K3

Mekk4 MAP3K4 Mtk1

Mekk5 MAP3K5 Ask1

Mekk6 MAP3K6 Ask2

Tak1 MAP3K7

Tpl-2 MAP3K8 Mekk8, Cot, Estf

Mlk1 MAP3K9 Mekk9,

Mlk2 MAP3K10 Mekk10, Mst

Mlk3 MAP3K11 Sprk, Ptk1

Zpk MAP3K12

lzk MAP3K13 Mekk13

niK MAP3K14

Ask3 MAP3K15 Mekk5-like

TAo1 MAP3K16 TAoK1, PSK2, MARKK

TAo2 MAP3K17 TAoK2, PSK

TAo3 MAP3K18 TAoK3, DPK, JiK

MlK7 ZAK AZK, MRK, MlTK


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Mammalian signaling has similar complexities due to exten-sive crosstalk between upstream activators, and there have been many observations that a single MAP3K can regulate multiple MAPK cascades. However, because kinases themselves can act as binding hubs, tethering functions in mammalian MAPK cascades can be shared by both core pathway components as well as dedicated scaffolding proteins. Most interactions with MAPKs are mediated by two short sequence motifs: the dock-ing (D or DEJL) motif and the FXF (DEF) motif, with one or more of these often present in scaffolds, activators, certain phosphatases, and many substrates.85,86

JNK-interacting protein (JIP) scaffolds, which organize JNK and sometimes p38 MAPK pathways, display some functional parallels to yeast Ste5. In a slight departure from the yeast situ-ation, several MAP3Ks contain docking sites that promote sta-ble interactions with specific MAPKs, such as the case with MEKK1 being able to bind tightly to JNKs through a docking motif.87 Indeed, the scaffolding functions of kinases cannot be overstated, especially since some dedicated scaffolds are pseudokinases whose tethering functions were likely selected over enzymatic ones during the course of evolution.83 The best-known example of this is the scaffold kinase suppressor of Ras (KSR), which was discovered in the sevenless eye development pathway in Drosophila and in the vulval induction pathway of C. elegans.88,89 Although KSR proteins are members of the Raf family, they lack the essential ATP-binding lysine residue required for functional kinase activity. In mammals KSR1 and KSR2 bind ERK1/2 and MEK1/2, and can allosterically activate Raf.43 Other proteins that influence assembly and activation of MAPK pathways include Sur8, CNK, MP-1 and IMP.86,90-92

Activation of MAPKs from the cell surface

Tyrosine kinase receptor activation of ERK1/2ERK1/2 are activated by a wide variety of stimuli that act through multiple cell surface receptors, and of these recep-tor tyrosine kinase (RTK) pathways are among the best

delin eated.14,18,34 Extracellular ligand binding triggers homo- and/or heterodimerization of RTKs, increasing kinase activ-ity and generating multiple phosphotyrosine motifs on RTKs and their dimerization partners. These motifs are recognized by SH2 domains on a variety of factors including the adap-tor proteins Shc and Grb2, which also contain proline-rich regions that can interact with the SH3 domain of the Ras guanine nucleotide exchange factor (GEF) son of sevenless (SOS). Upon association with the receptor-adaptor protein complex, SOS stimulates the exchange of GDP for GTP on Ras, promoting its interactions with a number of downstream effec-tors that include Raf. The direct interaction of Raf isoforms with Ras localizes these MAP3Ks to the plasma membrane and maintains proximity to non-receptor kinases, such as Src family members, p21-activated kinases (PAKs), and protein kinase C (PKC) isoforms, which in turn are able to phosphory-late Ras-Raf isoforms, altering their activity towards particu-lar substrates or enhancing interactions with other signaling factors.93,94 As discussed above, activated Raf isoforms can stimulate the MEK1/2-ERK1/2 pathway, thus forming the core MAPK module.

Activation of MAPKs by G-protein-coupled receptorsMany hormones act through G-protein-coupled receptors (GPCRs) to increase ERK1/2 activity, and as with RTK medi-ated stimulation, activation occurs primarily through Raf. Unlike RTK signaling to ERK1/2, however, GPCRs largely cir-cumvent Ras and instead utilize a variety of mechanisms to regulate Raf activity.

Figure 4 | JNK and ERK inhibitors

O

N NH

SP 600125 (1496)Selective JNK inhibitor

NN

NNH

H2NN

N

FR 180204 (3706)Selective ERK inhibitor

N N

HNO

NH

Cl

HO

HN

Cl

F

TCS ERK 11e (4465)Potent and selective ERK2 inhibitor

N

N

NN

O

HN

O

N

O

N

N

ERK5-IN-1 (5393)Potent and selective ERK5 inhibitor

Figure 3 | Regulation by Scaffolding

Sho11

Ste11

Ste7

Fus3

Kss1

Pbs2

Hog1p

Ste11

Ste5Ste5


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Figure 5 | p38 MAPK inhibitors

N

HN

N

F

SMe

O N

N

NN

OMe

OH

F

SB 203580 (1402)Selective inhibitor of p38 MAPK

SB 239063 (1962)Potent, selective p38 MAPK inhibitor;

orally active

N

O

O

NMe2

N

O

F

Cl

NN N

O

S

F

F

ClClSX 011 (3639)p38 MAPK inhibitor

VX 745 (3915)Potent and selective p38α inhibitor

Agonist binding to Gαs-coupled receptors results in activation of adenyl cyclase, which in turn raises the concentration of cyclic adenosine monophosphate (cAMP). While changes in cAMP concentration are known to affect ERK1/2 activity dif-ferentially, the pathway response appears cell-type specific or otherwise highly context-dependent.95,96 An increase in cAMP stimulates protein kinase A (PKA), a signaling molecule whose activities employ extensive cross-talk with ERK1/2 signaling.97 Inhibition of ERK1/2 activity is thought to involve phosphor-ylation of serine 41 and serine 621 on c-Raf by PKA, disturbing Raf associations with Ras and 14-3-3 protein, respectively.98,99 Conversely, PKA can also augment ERK1/2 sensitivity, amplitude, and duration.97

Gαi-coupled receptors can also regulate ERK1/2 signaling. Early in vivo evidence indicated that Gαi-coupled receptors most likely employ βγ subunits 100,101, and indeed, recent work on GPCR regulation of ERK1/2 has identified a novel mode of signaling that results in an alternative phosphorylation event that plays a causative role in cardiac hypertrophy.63

Inhibition of MAPK pathways

MAPK function studied by inhibitionMuch has been learned about MAPK signaling pathways through loss-of-function experiments utilizing dominant-neg-ative mutants, RNA interference, and small molecule inhibi-tors. However, because MAPKs are activated by overlapping upstream components and often share common substrates, the

dominant negative approach can become confused owing to inhibition of more than one target pathway. Similarly, gene-silencing approaches rely heavily on rescue experiments to determine whether a phenotype is pathway-dependent or due to an off-target effect and may require targeting multiple closely related enzymes. In view of these considerations, phar-macological inhibitors of MAPK pathway components present both a viable alternative and a complementary tool in under-standing the functional requirements of a given pathway.102,103 Although many inhibitors bind to the ATP binding pocket common to all protein kinases, the success of more specific allosteric inhibitors has spurred the search for molecules that bind to alternative pockets on kinase surfaces.104 MAPK path-way inhibitors are being developed that block essential protein- protein interactions, reflecting an improved understanding of how kinase signaling networks function.

ERK1/2 pathway inhibitorsOwing to the high degree of feedback control exhibited by the Raf-MEK-ERK pathway, direct inhibition of ERK1/2 is an attractive strategy to be used in conjunction with other inhibi-tors of upstream factors. The small molecule FR 180204 com-petes for the ATP-binding pocket of ERK1/2, with IC50 values of 0.31 and 0.14 μM, respectively, but its practical use is difficult to ascertain due to its targeting strategy.105 In contrast, the allos-teric inhibitor SCH772984 appears to both exquisitely inhibit the activity of ERK1/2 (with IC50 values of 0.004 and 0.001 μM, respectively) and prevent reactivation by MEK1/2, possibly by inducing a conformational change rendering ERK1/2 refrac-tory to feedback-regulated stimulation.106 Somewhat related to this is the inhibitor DEL-22379, which targets and interrupts ERK dimerization instead of its catalytic activity.69

In the mid 1990s, the MEK1/2 inhibitors PD 98059 and U0126 became available, allowing researchers to effectively interfere with the ERK1/2 pathway.107 PD 98059 was discovered through an in vitro kinase activation assay, while U0126 was identified in a cell-based assay as an inhibitor of AP-1 transcriptional activity.104,108,109 Both drugs are useful at low micromolar concentrations and inhibit activation of MEK1/2, but require higher concentrations to block already activated MEK1/2 in cells.103 U0126 and PD 98059 bind outside of the ATP bind-ing site of MEK1/2 and select for a low activity conformation, thereby shifting and maintaining the protein in an inactive state. Because of their binding mode, these drugs are among the most selective inhibitors available, with the only other kinase affected being the related MAP2K MEK5, which is inhibited at only slightly higher concentrations than MEK1/2. Subsequently, a number of MEK1/2 inhibitors have been devel-oped that are much more potent and display variable selectivity toward MEK5, including PD 198306, PD 0325901, and ARRY-142886 (AZD6244).110-113 PD 0325901 inhibits MEK1/2 activa-tion of ERK1/2 in cells at concentrations as low as 25 nM, but fails to inhibit a large panel of other protein kinases at more than 100 times the concentration.103 However, consistent with a ~10-fold greater potency towards MEK1/2 than MEK5, ERK5


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activation is inhibited at low micromolar concentrations by PD 035901 (for more on ERK5 inhibition, see Chen et al).114 The success of allosteric inhibitors of MEK1/2 has extended past the laboratory with several MEK1/2 targeting compounds currently in development as therapeutics.110,113,115-119

Another expanding group of compounds able to block the ERK1/2 pathway is directed against Raf isoforms. B-Raf in particular has emerged as an interesting target due to the high prevalence of B-Raf V600E mutations in melanomas. Several compounds have been identified that predominantly interact with this mutant form, including vemurafenib, encorafenib, PLX-4720, dabrafenib, and GDC-0879.120-124 Other drugs that target Raf are less selective, with compounds like Sorafenib, RO5126766, and AZ 628 inhibiting multiple kinases.125-127 Similarly, LY3009120, RAF265 (CHIR-265), TAK-632, ZM 336372, SB 590885 and GW5074 largely affect multiple isoforms of Raf.118,128-133

JNK pathway inhibitors SP 600125 is the most frequently used inhibitor of the JNK signaling pathway and blocks JNKs at concentrations in the range of 50-100 nM, however, this compound also inhibits several other protein kinases with roughly equal potency.134 CEP 1347 (KT-7515) blocks the JNK pathway, but is specifically an inhibitor of the upstream mixed lineage kinases (MLKs), so does not block JNKs that are regulated in an MLK-independent manner.135

p38 pathway inhibitors Several p38 inhibitors including SB 203580 have been devel-oped using pyridinyl imidazoles as lead compounds.136,137 Crystallographic studies indicate that SB 203580 prevents ATP binding by interacting with the active site of p38, but only for the α and β isoforms and not p38 γ and δ.138 A key feature of the ATP binding pocket that impacts inhibitor selectivity is the gatekeeper residue, which is a threonine residue at position

106 for p38 α and β.139 The smaller size of the gatekeeper (as opposed to the glutamine and methionine side chains present in p38 γ and δ) allows for accommodation of larger compounds in the active site.140 Consequently, Raf isoforms, due to their small gatekeeper side chains, can also interact with some p38 inhibitors.141 BIRB 796, a diaryl urea compound that is struc-turally unrelated to SB 203580, inhibits all four p38 isoforms by indirectly competing with the binding of ATP.142 There are several p38 pathway inhibitors being developed as therapeutics, including ralimetinib (LY 2228820), pexmetinib (ARRY 614) and losmapimod.143-149

Future prospectsThe last several years have seen an increased awareness that protein kinases are more than phosphorylating enzymes; they are multifunctional molecules evolved to process critical biological information. Given the prominent roles of MAPK pathways in both normal signal transmission and a variety of disease states, examination of these core modules will con-tinue to inform the general understanding of cellular signal-ing. MAPK networks rely on multiple kinases behaving in a multitude of ways, creating feedback loops and redundancies, switches and oscillators, and harnessing the activities of non-kinase proteins to give rise to robust signaling. The evolution-ary success of these pathways is rooted in their adaptability and versatility, and while much has been learned about the roles of these pathways in specific biological processes, current research continues to focus on the spatio-temporal dynam-ics underlying context-dependent signaling. As microscopy, mass spectrometry, and deep sequencing techniques continue to improve, the scale of focus will continue to shift away from individual pathways onto entire signaling networks. In partic-ular, pharmacological manipulation continues to be a crucial tool for assessing the functionality of MAPK signaling, paving the way for improved therapeutic strategies targeting numerous aspects of these multifaceted networks.


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References


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Target Cat No Name Description

MAP3K

ASK 5641 MSC 2032964A Potent and selective ASK1 inhibitor; orally bioavailable

4429 nQDi 1 ASK1 inhibitor

4825 TC ASK 10 Potent and selective ASK1 inhibitor; orally bioavailable

Raf 4836 AZ 628 Potent Raf inhibitor

4453 GDC 0879 Potent B-Raf inhibitor

1381 Gw 5074 Potent, selective c-Raf1 inhibitor

5260 KG 5 PDGFRβ, B-Raf, c-Raf, FlT3 and KiT inhibitor

5475 Kobe 0065 h-Ras-cRaf1 interaction inhibitor; inhibits Raf signaling

5036 Ml 786 Potent Raf inhibitor; orally bioavailable

1321 ZM 336372 Potent, selective c-Raf inhibitor

TAK 3604 (5Z)-7-oxozeaenol Potent and selective TAK1 MAPKKK inhibitor

5429 nG 25 TGF-β-activated kinase (TAK1) inhibitor

MAP2K (MEK)

1777 Arctigenin Potent MEK1 inhibitor; also inhibits iκBα phosphorylation

4842 BiX 02189 Selective MEK5 and ERK5 inhibitor

1532 10Z-hymenialdisine Pan kinase inhibitor; potently inhibits MEK1

4192 PD 0325901 Potent inhibitor of MEK1/2

4237 PD 184352 Selective MEK inhibitor

2605 PD 198306 Selective inhibitor of MEK1/2

4824 PD 334581 MEK1 inhibitor

1213 PD 98059 MEK inhibitor

1969 Sl 327 Selective inhibitor of MEK1 and MEK2; brain penetrant

1868 U0124 inactive analog of U0126 (Cat. no. 1144)

1144 U0126 Potent, selective inhibitor of MEK1 and 2

MAPK

ERK 5843 AX 15836 Potent and selective ERK5 inhibitor

4842 BiX 02189 Selective MEK5 and ERK5 inhibitor

5774 DEl 22379 ERK dimerization inhibitor

5393 ERK5-in-1 Potent and selective ERK5 inhibitor

3706 FR 180204 Selective ERK inhibitor

4433 Pluripotin Dual ERK1/RasGAP inhibitor; maintains ESC self-renewal

4465 TCS ERK 11e Potent and selective ERK2 inhibitor

3675 TMCB Dual-kinase inhibitor; inhibits CK2 and ERK8

4132 XMD 8-92 ERK5/BMK1 inhibitor; also BRD4 inhibitor

JNK 2651 AEG 3482 inhibitor of JnK signaling

3314 Bi 78D3 Selective, competitive JnK inhibitor

4924 CEP 1347 inhibitor of JnK signaling

5575 iQ 1S JnK inhibitor; anti-inflammatory

4550 iQ 3 Selective JnK3 inhibitor

1565 JiP-1 (153-163) JnK-selective inhibitor peptide

1496 SP 600125 Selective JnK inhibitor

5044 SR 3576 highly potent and selective JnK3 inhibitor

3607 SU 3327 Selective JnK inhibitor

2827 TCS JnK 5a Selective inhibitor of JnK2 and JnK3

3222 TCS JnK 6o Selective JnK inhibitor

p38 4753 Al 8697 Potent and selective p38α inhibitor

3920 AMG 548 Potent and selective p38α inhibitor

2186 CMPD-1 non-ATP-competitive p38α inhibitor; also tubulin polymerization inhibitor

5095 DBM 1285 p38 MAPK inhibitor; anti-inflammatory

Compounds Available from Tocris


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2908 Eo 1428 Selective inhibitor of p38α and p38β2

2657 JX 401 Potent, reversible p38α inhibitor

4586 Ml 3403 p38 inhibitor

2999 RwJ 67657 Potent, selective p38α and p38β inhibitor

1264 SB 202190 Potent, selective inhibitor of p38 MAPK

1202 SB 203580 Selective inhibitor of p38 MAPK

1402 SB 203580 hydrochloride Selective inhibitor of p38 MAPK; water-soluble

1962 SB 239063 Potent, selective p38 MAPK inhibitor; orally active

5040 SB 706504 p38 MAPK inhibitor

3528 SCio 469 Selective p38 MAPK inhibitor

2008 SKF 86002 p38 MAPK inhibitor; anti-inflammatory agent

3639 SX 011 p38 MAPK inhibitor

4254 TAK 715 Potent p38 MAPK inhibitor; anti-inflammatory

3916 VX 702 orally active p38α and p38β inhibitor

3915 VX 745 Potent and selective p38α inhibitor

Upstream and Receptor Signaling

EGFR 5318 AEE 788 Potent EGFR and VEGFR inhibitor

1276 AG 1478 highly potent EGFR-kinase inhibitor

0493 AG 18 EGFR/PDGFR-kinase inhibitor

0414 AG 490 EGFR-kinase inhibitor; also JAK2, JAK3 inhibitor

0618 AG 555 Potent EGFR-kinase inhibitor

0616 AG 556 EGFR-kinase inhibitor

1555 AG 825 Selective ErbB2 inhibitor

2617 AG 879 ErbB2 inhibitor; also inhibits TrkA and VEGFR-2

0497 AG 99 EGFR-kinase inhibitor

2417 BiBU 1361 Selective inhibitor of EGFR-kinase

2416 BiBX 1382 highly selective EGFR-kinase inhibitor

5022 BMS 599626 Potent, selective EGFR and ErbB2 inhibitor

3360 CGP 52411 EGFR inhibitor; also inhibits Aβ42 fibril formation

4502 DiM inhibits phosphorylation of EGFR and downstream activation of ERK; also activates Chk2

1110 Genistein EGFR kinase inhibitor; also estrogen and PPARγ ligand

2239 Gw 583340 Potent dual EGFR/ErbB2 inhibitor; orally active

2646 hDS 029 Potent inhibitor of the ErbB receptor family

3580 hKi 357 Dual irreversible inhibitor of ErbB2 and EGFR

3000 iressa orally active, selective EGFR inhibitor

3352 JnJ 28871063 Potent ErbB receptor family inhibitor

1331 lavendustin A EGFR, p60c-src inhibitor

1037 PD 153035 EGFR kinase inhibitor

2615 PD 158780 Potent ErbB receptor family inhibitor

4941 PKi 166 Potent EGFR-kinase inhibitor

3599 TAK 165 Potent and selective ErbB2 inhibitor

3115 whi-P 154 JAK3 kinase inhibitor; also inhibits EGFR

FAK 3414 FAK inhibitor 14 Selective FAK inhibitor

4278 PF 431396 Dual FAK/PyK2 inhibitor

3239 PF 573228 Potent and selective FAK inhibitor

4498 y 11 Potent and selective FAK inhibitor

FTI (Ras) 5424 Deltarasin high affinity PDEδ-KRAS interaction inhibitor

2407 FTi 277 inhibits h-Ras and K-Ras processing; prodrug form of FTi 276 (Cat. no. 2406)

5607 GnF 7 Ras signaling inhibitor; inhibits Ack1 and GCK

4989 Salirasib Ras inhibitor; also induces autophagy

3101 XRP44X Ras-net (Elk-3) pathway inhibitor


tocris.com | 11


IRAK 5665 iRAK 1/4 inhibitor i iRAK4 and iRAK1 inhibitor

PI3-K 1983 740 y-P Cell-permeable Pi 3-kinase activator

4839 AZD 6482 Potent and selective Pi 3-Kβ inhibitor

4674 CZC 24832 Selective inhibitor of Pi 3-kinase γ

4840 KU 0060648 Dual DnA-PK and Pi 3-K inhibitor

1130 ly 294002 Prototypical Pi 3-kinase inhibitor; also inhibits other kinases

2418 ly 303511 negative control of ly 294002 (Cat. no. 1130)

3977 3-Methyladenine Class iii Pi 3-kinase inhibitor; also inhibits autophagy

4820 PF 04691502 Potent and selective dual Pi 3-K/mToR inhibitor

2930 Pi 103 hydrochloride inhibitor of Pi 3-kinase, mToR and DnA-PK

1125 Quercetin non-selective Pi 3-kinase inhibitor

1232 wortmannin Potent, irreversible inhibitor of Pi 3-kinase; also inhibitor of PlK1

VEGFR/PDGFR 5013 AC 710 Potent and selective PDGFR family inhibitor

4274 AP 24534 Potent VEGFR, PDGFR and FGFR inhibitor; also pan-Bcr-Abl inhibitor

4350 Axitinib Potent VEGFR-1, -2 and -3 inhibitor

4471 DMh4 Selective VEGFR-2 inhibitor

1222 DMPQ Potent, selective inhibitor of PDGFRβ

4931 EG 00229 neuropilin 1 (nRP1) receptor antagonist; inhibits VEGFA binding to nRP1

5260 KG 5 PDGFRβ, B-Raf, c-Raf, FlT3 and KiT inhibitor

2542 Ki 8751 Potent, selective VEGFR-2 inhibitor

3785 PD 166285 Potent Src inhibitor; also inhibits FGFR1, PDGFRβ and wee1

3304 SU 16f Potent and selective PDGFRβ inhibitor

1459 SU 4312 Potent inhibitor of VEGFR tyrosine kinase

3300 SU 5402 Potent FGFR and VEGFR inhibitor

3037 SU 5416 VEGFR inhibitor; also inhibits KiT, RET, MET and FlT3

3335 SU 6668 PDGFR, VEGFR and FGFR inhibitor

3768 Sunitinib Potent VEGFR, PDGFRβ and KiT inhibitor

3909 Toceranib Potent PDGFR and VEGFR inhibitor

5680 Vatalanib Potent VEGFR inhibitor; also aromatase inhibitor

5422 Xl 184 Potent VEGFR inhibitor; also inhibits other RTKs

2499 ZM 306416 inhibitor of VEGF receptor tyrosine kinase

2475 ZM 323881 Potent, selective inhibitor of VEGFR-2

Regulators

14-3-3 2145 Difopein high affinity inhibitor of 14.3.3 proteins; induces apoptosis

2144 R18 inhibitor of 14.3.3 proteins

Akt 3897 APi-1 Selective Akt/PKB inhibitor; also antitumor

2151 APi-2 Selective inhibitor of Akt/PKB signaling; also antitumor and antiviral

2558 10-DEBC Selective Akt/PKB inhibitor

2926 FPA 124 Akt/PKB inhibitor

4144 GSK 690693 Akt kinase inhibitor; also antitumor

5682 oSU 03012 PDK1 inhibitor; inhibits Akt signaling

4598 PhT 427 Dual Akt and PDK1 inhibitor; antitumor

4398 SC 66 Allosteric Akt inhibitor

4635 SC 79 Akt activator

Hsp90 1515 17-AAG Selective hsp90 inhibitor

4608 BiiB 021 Selective hsp90 inhibitor

2435 CCT 018159 hsp90 inhibitor

2610 17-DMAG water-soluble hsp90 inhibitor

4701 EC 144 high affinity, potent and selective hsp90 inhibitor

3387 Gedunin hsp90 inhibitor; exhibits anticancer and antimalarial activity

1368 Geldanamycin Selective hsp90 inhibitor


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1629 herbimycin A hsp90 inhibitor; also Src family kinase inhibitor

5480 KRiBB11 hSF1 inhibitor

3061 Macbecin i hsp90 inhibitor

3104 PU h71 Potent hsp90 inhibitor

1589 Radicicol hsp90 inhibitor; antifungal antibiotic

PAK 5190 FRAX 486 Potent p21-activated kinase (PAK) inhibitor; brain penetrant and orally bioavailable

3622 iPA 3 Group i p21-activated kinase (PAK) inhibitor

4212 PiR 3.5 negative control of iPA 3 (Cat. no. 3622)

PKC 4128 Bisindolylmaleimide ii Potent PKC inhibitor and nicotinic receptor antagonist

2383 Bryostatin 1 Protein kinase C activator

5237 Bryostatin 2 Protein kinase C activator

1626 Calphostin C Potent, selective and photo-dependent PKC inhibitor

1330 Chelerythrine Cell-permeable protein kinase C inhibitor

0741 GF 109203X Protein kinase C inhibitor

2253 Go 6976 Potent protein kinase C inhibitor; selective for α and β isozymes

2285 Go 6983 Broad spectrum PKC inhibitor

4738 ly 333531 Protein kinase C inhibitor; selective for β isozymes

1193 Melittin PKC and cAMP-dependent protein kinases inhibitor; also inhibits Gs and stimulates Gi activity

4054 PEP 005 Protein kinase C activator

4153 Phorbol 12,13-dibutyrate Protein kinase C activator

1201 Phorbol 12-myristate 13-acetate

Protein kinase C activator

2992 PKC 412 Protein kinase C inhibitor

1791 PKC ζ pseudosubstrate PKC ζ inhibitor peptide (attached to cell-permeable vector)

5739 Prostratin PKC activator; also nF-κB activator

1790 Pseudo RACK1 Protein kinase C activator peptide (attached to cell permeable vector)

1610 Rottlerin Reported PKCδ inhibitor

2549 ZiP Cell-permeable inhibitor of atypical PKC isozyme PKMζ

PP2A 1336 Calyculin A Protein phosphatase 1 and 2A inhibitor

1548 Cantharidin Protein phosphatase 1 and 2A inhibitor

1840 Fostriecin sodium salt Potent PP2A and PP4 inhibitor

1136 okadaic acid Protein phosphatase 1 and 2A inhibitor

SGK 3572 GSK 650394 Serum- and glucocorticoid-regulated kinase (SGK) inhibitor

Src 3914 A 419259 inhibitor of Src family kinases

3963 AZM 475271 Src tyrosine kinase inhibitor

4361 Bosutinib Dual Src-Abl inhibitor; antiproliferative

4660 KB SRC 4 Potent and selective c-Src inhibitor

2265 lyn peptide inhibitor inhibits lyn-dependent activities of il-5 receptor; cell-permeable

4582 MlR 1023 Selective allosteric activator of lyn kinase

2877 MnS Selective inhibitor of Src and Syk

3063 1-naphthyl PP1 Src family kinase inhibitor; also inhibits c-Abl

3785 PD 166285 Potent Src inhibitor; also inhibits FGFR1, PDGFRβ and wee1

1554 Piceatannol p56lck and Syk kinase inhibitor; inhibits TnF-induced nF-κB activation

1397 PP 1 Potent, selective Src family kinase inhibitor

1407 PP 2 Potent, selective Src family kinase inhibitor

2794 PP 3 negative control for PP 2 (Cat. no. 1407)

4763 Pyridostatin Targets the proto-oncogene SRC; stabilizes G-quadruplexes

3642 Src i1 Dual site Src kinase inhibitor

3567 TC-S 7003 Potent lck inhibitor

5413 wh-4-023 Potent and selective lck and Src inhibitor; also inhibits SiK


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Downstream Effectors/Targets

AP-1 5309 SP 100030 nF-κB and AP-1 dual inhibitor

2476 SR 11302 AP-1 inhibitor; antitumor agent

JNK/c-Jun 1290 Anisomycin JnK, SAPK and p38 activator

1989 c-JUn peptide Peptide inhibitor of JnK/c-Jun interaction

MK2 4279 PF 3644022 Potent MK2 inhibitor

3140 PhA 767491 MK2 inhibitor; also inhibits cdc7/cdk9

Mnk 4500 Cercosporamide Potent Mnk2 inhibitor

2731 CGP 57380 Selective inhibitor of Mnk1

5183 ETP 45835 Mnk1 and Mnk2 inhibitor

MSK1 4630 SB 747651A Potent MSK1 inhibitor; also inhibits other AGC group kinases

NFAT 3930 nFAT inhibitor inhibitor of calcineurin-mediated nFAT activation

5710 nFAT inhibitor, Cell Permeable

Cell permeable nFAT inhibitor

RSK 4037 BRD 7389 Ribosomal S6 kinase inhibitor

3603 Kaempferol RSK2 inhibitor; also downregulates PlK1 expression and activates mitochondrial Ca2+ uniporter

4032 PF 4708671 S6K1 inhibitor

1292 Rapamycin inhibits p70 S6K activation; mToR inhibitor

2250 Sl 0101-1 Selective ribosomal S6 kinase (RSK) inhibitor


For a complete and up-to-date product listing please visit tocris.com

http://www.tocris.com


14 |

Tocriscreen Kinase Inhibitor Toolbox (3514)Inhibitors of the most important kinase targets all on one plate

80 kinase inhibitors pre-dissolved in DMSO 1 set (250 μl, 10 mM solutions)

The Kinase inhibitor Toolbox is a unique collection of 80 compounds from the Tocris catalog, many exclusive to us, covering a wide range of kinase targets, including multiple inhibitors of commonly studied pathway mediators, such as EGFR, MAPK, and AMPK cascades, among others.

For more information, visit: www.tocris.com/tocriscreen

Related literature from Tocris:

Kinases Product ListingA collection of over 400 products for kinase research, the listing includes inhibitors of:

• Receptor Tyrosine Kinases • Protein Kinases A, C, D and G• Pi-3 Kinase, Akt and mToR• MAPK Signaling• Receptor Serine/Threonine Kinases

Cancer Research Product GuideA collection of over 750 products for cancer research, the guide includes research tools for the study of:

• Cancer Metabolism• Receptor Signaling• Angiogenesis

Immunology Product ListingA collection of over 190 products for immunology research, the guide includes research tools for the study of:

• Chemokine and Cytokine Signaling• Chemotaxis• Complement System• immune Cell Signaling• inflammation

To download or request a copy please visit: www.tocris.com/requestliterature

• Epigenetics in Cancer• Cell Cycle and DnA Damage Repair• invasion and Metastasis

Citations:Moujalled et al (2013) Kinase inhibitor screening identifies cyclin-dependent kinases and glycogen synthase kinase 3 as potential modulators of TDP-43 cytosolic accumulation during cell stress. PLoS One 8 e67433. PMiD: 23840699.Jester et al (2010) A coiled-coil enabled split-luciferase three-hybrid system: applied toward profiling inhibitors of protein kinases. J.Am.Chem.Soc. 132 11727. PMiD: 20669947.

Also coming soon: Kinase Inhibitor Toolbox II

http://www.tocris.com/tocriscreen

http://www.tocris.com/requestliterature


tocris.com | 15

Molecule Catalog Number Species Clonality Antibody Applications

4EBP1 AF3227 human/Mouse Polyclonal Antibody Sw, wB

c-Myc MAB3696 human Monoclonal Antibody FC, iCC/iF, ihC, iP, wB

eiF4E MAB3228 human/Mouse/Rat Monoclonal Antibody iCC/iF, wB

ERK1/ERK2 (T202/y204, T185/y187) MAB1018 human/Mouse/Rat Monoclonal Antibody iCC/iF, FC, Sw, wB

p70 S6 Kinase AF8962 human/Mouse/Rat Polyclonal Antibody ihC, FC, Sw, wB

p70 S6 Kinase (T389) MAB8963 human Recombinant Monoclonal Antibody iCC/iF, wB

PlC-gamma 1 (y783) MAB74541 human/Mouse/Rat Recombinant Monoclonal Antibody wB

PlC-gamma 2 MAB3716 human/Mouse Monoclonal Antibody iCC/iF, wB

PlC-gamma 2(y759) MAB7377 human Monoclonal Antibody iCC/iF, wB

Rheb MAB3426 human/Mouse/Rat Monoclonal Antibody ihC, wB

Ribosomal Protein S6/RPS6 (S235/S236) AF3918 human/Mouse/Rat Polyclonal Antibody ihC, Sw, wB

RSK (Pan) MAB2056 human/Mouse/Rat Monoclonal Antibody Sw, wB

RSK1/RSK2 (S221, S227) MAB892 human/Mouse Recombinant Monoclonal Antibody Sw, wB

Src (y416) MAB2685 human Recombinant Monoclonal Antibody iCC/iF, Sw, wB

ToR AF15371 human/Mouse/Rat Polyclonal Antibody iP, wB

Application Key: FC FC, iCC/iF iCC/iF/immunofluorescence, ihC ihC, iP iP, Sw Simple western, wB western Blot

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MAPK Signaling Pathway: Mitogen Stimulation Pathway

View Interactive Pathway: rndsystems.com/pathways_mapkmitogen

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RasGRP1

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PLC gamma

Raf

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KSRERK1/2MEK1/2

MEK1/2

Src

p70 S6 Kinase

PDCD4

eIF4A

eIF4G

eIF4B

eIF4E

4EBP

mTORC1

Rheb

TSC2

TSC1

Sef

RSK

ERK1/2

ERK1/2MP1

p14

Endosome

ERK1/2UBF

RNA Pol ICAD

E2F

RB1

CDK4

Cyclin D

TOB

Myc

MEK1/2

Growth andproliferation

RNA, DNA,Phospholipids

rRNA

60S 40S

http://www.rndsystems.com/pathways_mapkmitogen

North America Tel: (800) 343 7475

[email protected] Tel: +86 (21) 52380373

Rest of World bio-techne.com/find-us/distributors Tel: +1 612 379 2956

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map kinase pathways: functions and modulation · 2017-05-25 · tocris scientific review series 2 |...

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